Osteocytes but not osteoblasts directly build mineralized bone structures.

Bone-forming osteoblasts have been a cornerstone of bone biology for more than a century. Most research toward bone biology and bone diseases center on osteoblasts. Overlooked are the 90% of bone cells, called osteocytes. This study aims to test the hypothesis that osteocytes but not osteoblasts directly build mineralized bone structures, and that defects in osteocytes lead to the onset of hypophosphatemia rickets. The hypothesis was tested by developing and modifying multiple imaging techniques, including both in vivo and in vitro models plus two types of hypophosphatemia rickets models (Dmp1-null and Hyp, Phex mutation mice), and Dmp1-Cre induced high level of ß-catenin models. Our key findings were that osteocytes (not osteoblasts) build bone similar to the construction of a high-rise building, with a wire mesh frame (i.e., osteocyte dendrites) and cement (mineral matrices secreted from osteocytes), which is a lengthy and slow process whose mineralization direction is from the inside toward the outside. When osteoblasts fail to differentiate into osteocytes but remain highly active in Dmp-1-null or Hyp mice, aberrant and poor bone mineralization occurs, caused by a sharp increase in Wnt-ß-catenin signaling. Further, the constitutive expression of ß-catenin in osteocytes recaptures a similar osteomalacia phenotype as shown in Dmp1 null or Hyp mice. Thus, we conclude that osteocytes directly build bone, and osteoblasts with a short life span serve as a precursor to osteocytes, which challenges the existing dogma.

Changes in the intra- and peri-cellular sclerostin distribution in lacuno-canalicular system induced by mechanical unloading.

INTRODUCTION: Mechanical stimuli regulate Sclerostin (Scl), a negative regulator of bone formation, expression in osteocytes. However, the detailed Scl distribution in osteocytes in response to mechanical unloading remains unclear. MATERIALS AND METHODS: Twelve-week-old male rats were used. The sciatic and femoral nerves on the right side were excised as mechanical unloading treatment. A sham operation was performed on the left side. One week after neurotrauma, the bone density of the femora was evaluated by peripheral quantitative computed tomography, and immunofluorescence was performed in coronal sections of the femoral diaphysis. The mean fluorescence intensity and fluorescent profile of Scl from the marrow to the periosteal side were analyzed to estimate the Scl expression and determine to which side (marrow or periosteal) the Scl prefers to distribute in response to mechanical unloading. The most sensitive region indicated by the immunofluorescence results was further investigated by transmission electron microscopy (TEM) with immunogold staining to show the Scl expression changes in different subcellular structures. RESULTS: In femur distal metaphysis, neurotrauma-induced mechanical unloading significantly decreased the bone density, made the distribution of Scl closer to the marrow on the anterior and medial side, and increased the Scl expression only on the lateral side. TEM findings showed that only the expression of Scl in canaliculi was increased by mechanical unloading. CONCLUSIONS: Our results showed that even short-term mechanical unloading is enough to decrease bone density, and mechanical unloading not only regulated the Scl expression but also changed the Scl distribution in both the osteocyte network and subcellular structures.

Ultrastructural analysis of different skeletal cell types in mucopolysaccharidosis dogs at the onset of postnatal growth.

The mucopolysaccharidoses (MPS) are a family of lysosomal storage disorders characterized by deficient activity of enzymes that degrade glycosaminoglycans (GAGs). Abnormal development of the vertebrae and long bones is a hallmark of skeletal disease in several MPS subtypes; however, the underlying cellular mechanisms remain poorly understood. The objective of this study was to conduct an ultrastructural examination of how lysosomal storage differentially affects major skeletal cell types in MPS I and VII using naturally occurring canine disease models. We showed that both bone and cartilage cells from MPS I and VII dog vertebrae exhibit significantly elevated storage from early in postnatal life, with storage generally greater in MPS VII than MPS I. Storage was most striking for vertebral osteocytes, occupying more than forty percent of cell area. Secondary to storage, dilation of the rough endoplasmic reticulum (ER), a marker of ER stress, was observed most markedly in MPS I epiphyseal chondrocytes. Significantly elevated immunostaining of light chain 3B (LC3B) in MPS VII epiphyseal chondrocytes suggested impaired autophagy, while significantly elevated apoptotic cell death in both MPS I and VII chondrocytes was also evident. The results of this study provide insights into how lysosomal storage differentially effects major skeletal cell types in MPS I and VII, and suggests a potential relationship between storage, ER stress, autophagy, and cell death in the pathogenesis of MPS skeletal defects.

Assessment of Osteocytes: Techniques for Studying Morphological and Molecular Changes Associated with Perilacunar/Canalicular Remodeling of the Bone Matrix.

Recent advances have revived interest in the concept of osteocyte perilacunar/canalicular remodeling (PLR) and have motivated efforts to identify the mechanisms regulating this process in bone in the context of normal physiology and pathological conditions. Here, we describe several methods that are evaluating morphological changes associated with PLR function of osteocytes.

The microgravity induces the ciliary shortening and an increased ratio of anterograde/retrograde intraflagellar transport of osteocytes.

It is hard to explain the decrease in mechanosensitivity of osteocytes under microgravity. Primary cilia are essential mechanosensor for osteocytes. The cilia become shorter under the simulated microgravity (SMG) environment. The cilia change may be the reason for the mechanosensitivity decrease of osteocytes under SMG. To reveal the role of primary cilia in weightless-induced osteocyte dysfunction, we investigate intraflagellar transport (IFT) to understand the mechanism of the decreased cilia length of osteocytes when subjected to SMG. We measure the number of anterograde IFT particles with GFP::IFT88 and retrograde IFT particles with OFP::IFT43 that occur at a particular transverse plane of the cilia. We also measure the expression of IFT88 and IFT43 and the size of IFT particles under SMG. Herein, the ratio of anterograde/retrograde particle number and the ratio of protein expression of IFT88/IFT43 increase under SMG. The size of anterograde IFT particles with GFP::IFT88 gets a significant decrease under SMG. Fundamentally, SMG has broken the balanced operating state of IFT and makes the IFT particles smaller. The phenomenon under SMG is intriguing.

Assessment of the human bone lacuno-canalicular network at the nanoscale and impact of spatial resolution.

Recently, increasing attention has been given to the study of osteocytes, the cells that are thought to play an important role in bone remodeling and in the mechanisms of bone fragility. The interconnected osteocyte system is deeply embedded inside the mineralized bone matrix and lies within a closely fitted porosity known as the lacuno-canalicular network. However, quantitative data on human samples remain scarce, mostly measured in 2D, and there are gaps to be filled in terms of spatial resolution. In this work, we present data on femoral samples from female donors imaged with isotropic 3D spatial resolution by magnified X-ray phase nano computerized-tomography. We report quantitative results on the 3D structure of canaliculi in human femoral bone imaged with a voxel size of 30 nm. We found that the lacuno-canalicular porosity occupies on average 1.45% of the total tissue volume, the ratio of the canalicular versus lacunar porosity is about 37.7%, and the primary number of canaliculi stemming from each lacuna is 79 on average. The examination of this number at different distances from the surface of the lacunae demonstrates branching in the canaliculi network. We analyzed the impact of spatial resolution on quantification by comparing parameters extracted from the same samples imaged with 120 nm and 30 nm voxel sizes. To avoid any bias related to the analysis region, the volumes at 120 nm and 30 nm were registered and cropped to the same field of view. Our results show that the measurements at 120 and 30 nm are strongly correlated in our data set but that the highest spatial resolution provides more accurate information on the canaliculi network and its branching properties.

Graphene Oxide Enhances Chitosan-Based 3D Scaffold Properties for Bone Tissue Engineering.

The main goal of bone tissue engineering (BTE) is to refine and repair major bone defects based on bioactive biomaterials with distinct properties that can induce and support bone tissue formation. Graphene and its derivatives, such as graphene oxide (GO), display optimal properties for BTE, being able to support cell growth and proliferation, cell attachment, and cytoskeleton development as well as the activation of osteogenesis and bone development pathways. Conversely, the presence of GO within a polymer matrix produces favorable changes to scaffold morphologies that facilitate cell attachment and migration i.e., more ordered morphologies, greater surface area, and higher total porosity. Therefore, there is a need to explore the potential of GO for tissue engineering applications and regenerative medicine. Here, we aim to promote one novel scaffold based on a natural compound of chitosan, improved with 3 wt.% GO, for BTE approaches, considering its good biocompatibility, remarkable 3D characteristics, and ability to support stem cell differentiation processes towards the bone lineage.

Exosomes derived from Wharton's jelly of human umbilical cord mesenchymal stem cells reduce osteocyte apoptosis in glucocorticoid-induced osteonecrosis of the femoral head in rats via the miR-21-PTEN-AKT signalling pathway.

Purpose: Glucocorticoid-induced osteonecrosis of the femoral head (GIONFH) is a common disease after long-term or high-dose glucocorticoid use. The pathogenesis of GIONFH is still controversial, and abnormal bone metabolism caused by glucocorticoids may be one of the important factors. Exosomes, owing to their positive effect on bone repair, show promising therapeutic effects on bone-related diseases. In this study, we hypothesised that exosomes reduce osteocyte apoptosis in rat GIONFH via the miR-21-PTEN-AKT signalling pathway. Methods: To evaluate the effects of exosomes in GIONFH, a dexamethasone-treated or exosome-treated in vitro cell model and a methylprednisolone-treated in vivo rat model were set up. In vitro, a CCK-8 assay and 5-ethynyl-2'-deoxyuridine staining were performed to evaluate the proliferation of osteocytes. Further, a terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay, annexin V-fluorescein isothiocyanate-propidium iodide staining, and western blotting were conducted to evaluate the apoptosis of osteocytes. In vivo, we used micro-computed tomography and histological and immunohistochemical analyses to assess the effects of exosomes. Moreover, the mechanism of exosome action on osteocyte apoptosis through the miR-21-PTEN-AKT pathway was investigated by high-throughput RNA sequencing, fluorescence in situ hybridisation, luciferase reporter assays, and western blotting. Results: High-throughput RNA sequencing results showed that the AKT signalling pathway was up-regulated in the exosome group. Quantitative PCR and western blotting confirmed that the relative expression of genes in the AKT pathway was up-regulated. Western blotting revealed that AKT activated by exosomes inhibited osteocyte apoptosis. RNA fluorescence in situ hybridisation and luciferase reporter assays were performed to confirm the interaction between miR-21 and PTEN. According to the experiment in vivo, exosomes prevented GIONFH in a rat model as evidenced by micro-computed tomography scanning and histological and immunohistochemical analyses. Conclusions: Exosomes are effective at inhibiting osteocyte apoptosis (in MLO-Y4 cells) and at preventing rat GIONFH. These beneficial effects are mediated by the miR-21-PTEN-AKT signalling pathway.

Microenvironment engineering of osteoblastic bone metastases reveals osteomimicry of patient-derived prostate cancer xenografts.

Representative in vitro models that mimic the native bone tumor microenvironment are warranted to support the development of more successful treatments for bone metastases. Here, we have developed a primary cell 3D model consisting of a human osteoblast-derived tissue-engineered construct (hOTEC) indirectly co-cultured with patient-derived prostate cancer xenografts (PDXs), in order to study molecular interactions in a patient-derived microenvironment context. The engineered biomimetic microenvironment had high mineralization and embedded osteocytes, and supported a high degree of cancer cell osteomimicry at the gene, protein and mineralization levels when co-cultured with prostate cancer PDXs from a lymph node metastasis (LuCaP35) and bone metastasis (BM18) from patients with primary prostate cancer. This fully patient-derived model is a promising tool for the assessment of new molecular mechanisms and as a personalized pre-clinical platform for therapy testing for patients with prostate cancer bone metastases.

Aberrant activation of Wnt signaling pathway altered osteocyte mineralization.

Mineralization of bone is a dynamic process, involving a complex interplay between cells, secreted macromolecules, signaling pathways, and enzymatic reactions; the dysregulation of bone mineralization may lead to serious skeletal disorders, including hypophosphatemic rickets, osteoporosis, and rheumatoid arthritis. Very few studies have reported the role of osteocytes - the most abundant bone cells in the skeletal system and the major orchestrators of bone remodeling in bone mineralization, which is owed to their nature of being deeply embedded in the mineralized bone matrix. The Wnt/ß-catenin signaling pathway is actively involved in various life processes including osteogenesis; however, the role of Wnt/ß-catenin signaling in the terminal mineralization of bone, especially in the regulation of osteocytes, is largely unknown. This research demonstrates that during the terminal mineralization process, the Wnt/ß-catenin pathway is downregulated, and when Wnt/ß-catenin signaling is activated in osteocytes, dendrite development is suppressed and the expression of dentin matrix protein 1 (DMP1) is inhibited. Aberrant activation of Wnt/ß-catenin signaling in osteocytes leads to the spontaneous deposition of extra-large mineralized nodules on the surface of collagen fibrils. The altered mineral crystal structure and decreased bonding force between minerals and the organic matrix indicate the inferior integration of minerals and collagen. In conclusion, Wnt/ß-catenin signaling plays a critical role in the terminal differentiation of osteocytes and as such, targeting Wnt/ß-catenin signaling in osteocytes may serve as a potential therapeutic approach for the management of bone-related diseases.

No Signature of Osteocytic Osteolysis in Cortical Bone from Lactating NMRI Mice.

The roles of osteocytes in bone homeostasis have garnered increasing attention since it has been realized that osteocytes communicate with other organs. It has long been debated whether and/or to which degree osteocytes can break down the bone matrix surrounding them in a process called osteocytic osteolysis. Osteocytic osteolysis has been indicated to be induced by a number of skeletal challenges including lactation in CD1 and C57BL/6 mice, whereas immobilization-induced osteocytic osteolysis is still a matter of controversy. Motivated by the wish to understand this process better, we studied osteocyte lacunae in lactating NMRI mice, which is a widely used outbred mouse strain. Surprisingly, no trace of osteocytic osteolysis could be detected in tibial or femoral cortical bone either by 3D investigation by synchrotron nanotomography, by studies of lacunar cross-sectional areas using scanning electron microscopy, or by light microscopy. These results lead us to conclude that osteocytic osteolysis does not occur in NMRI mice as a response to lactation, in turn suggesting that osteocytic osteolysis may not play a generic role in mobilizing calcium during lactation.

Investigating Osteocytic Perilacunar/Canalicular Remodeling.

PURPOSE OF REVIEW: In perilacunar/canalicular remodeling (PLR), osteocytes dynamically resorb, and then replace, the organic and mineral components of the pericellular extracellular matrix. Given the enormous surface area of the osteocyte lacuna-canalicular network (LCN), PLR is important for maintaining homeostasis of the skeleton. The goal of this review is to examine the motivations and critical considerations for the analysis of PLR, in both in vitro and in vivo systems. RECENT FINDINGS: Morphological approaches alone are insufficient to elucidate the complex mechanisms regulating PLR in the healthy skeleton and in disease. Understanding the role and regulation of PLR will require the incorporation of standardized PLR outcomes as a routine part of skeletal phenotyping, as well as the development of improved molecular and cellular outcomes. Current PLR outcomes assess PLR enzyme expression, the LCN, and bone matrix composition and organization, among others. Here, we discuss current PLR outcomes and how they have been applied to study PLR induction and suppression in vitro and in vivo. Given the role of PLR in skeletal health and disease, integrated analysis of PLR has potential to elucidate new mechanisms by which osteocytes participate in skeletal health and disease.

Uranium Effect on Osteocytic Cells In Vitro.

Once absorbed in the body, natural uranium [U(VI)], a radionucleotide naturally present in the environment, is targeted to the skeleton which is the long-term storage organ. We and others have reported the U(VI) negative effects on osteoblasts (OB) and osteoclasts (OC), the main two cell types involved in bone remodeling. In the present work, we addressed the U(VI) effect on osteocytes (OST), the longest living bone cell type and the more numerous (> 90%). These cells, which are embedded in bone matrix and thus are the more prone to U(VI) long-term exposure, are now considered as the chief orchestrators of the bone remodeling process. Our results show that the cytotoxicity index of OST is close to 730 µM, which is about twice the one reported for OB and OC. However, despite this resistance potential, we observed that chronic U(VI) exposure as low as 5 µM led to a drastic decrease of the OST mineralization function. Gene expression analysis showed that this impairment could potentially be linked to an altered differentiation process of these cells. We also observed that U(VI) was able to trigger autophagy, a highly conserved survival mechanism. Extended X-ray absorption fine structure analysis at the U LIII edge of OST cells exposed to U(VI) unambiguously shows the formation of an uranyl phosphate phase in which the uranyl local structure is similar to the one present in Autunite. Thus, our results demonstrate for the first time that OST mineralization function can be affected by U(VI) exposure as low as 5 µM, suggesting that prolonged exposure could alter the central role of these cells in the bone environment.

Towards a Connectomic Description of the Osteocyte Lacunocanalicular Network in Bone.

PURPOSE OF REVIEW: Osteocytes are the most abundant bone cells. They are completely encased in mineralized tissue, sitting inside lacunae that are connected by a multitude of canaliculi. In recent years, the osteocyte network has been shown to fulfill endocrine functions and to communicate with a number of other organs. This review addresses emerging knowledge on the connectome of the lacunocanalicular network in different types of bone tissue. RECENT FINDINGS: Recent advances in three-dimensional imaging technology started to reveal parameters that are well known from general theory to characterize the function of networks, such as network density, degree of nodes, or shortest path length through the network. The connectome of the lacunocanalicular network differs in some aspects between lamellar and woven bone and seems to change with age. More research is needed to relate network structure to function, such as intercellular transport or communication and its role in mechanosensation, as well as to understand the effect of diseases.

New insights into the process of osteogenesis of anosteocytic bone.

The bone material of almost all vertebrates contains the same cellular components. These comprise osteoblasts that produce bone, osteoclasts that resorb bone and osteocytes, which are the master regulators of bone metabolism, particularly bone modeling and remodeling. It is thus surprising that the largest group of extant vertebrates, neoteleost fish, lacks osteocytes entirely (anosteocytic bone). Osteocytes are the progeny of osteoblasts, which become entrapped in the osteoid they secrete, then undergo several morphologic and functional changes, to finally form an intricate network of living cells in the bone matrix. While the process of osteogenesis of osteocytic bone has been thoroughly studied, osteogenesis of anosteocytic bone is less well understood. The current paradigm for formation of anosteocytic bone suggests that osteoblasts remain always on the external surface of the formed bone, and do not become entrapped in the osteoid. Such a process requires the osteoblasts to function in a fundamentally-different way from osteoblasts of all other bony vertebrates. Here we present a comparative structural study of the osteocytic bones of zebrafish and anosteocytic bones of medaka and show that they are remarkably similar in structure at several hierarchical levels. Scanning electron microscopy and phase contrast-enhanced µCT reveal the presence of numerous mineralized objects in the matrix of anosteocytic bone. These objects resemble osteocytic lacunae in zebrafish bone, and their locations and distribution are similar to those of osteocytes in zebrafish bone. Our findings provide support for the occurrence of a process of anosteocytic bone osteogenesis that has so far been rejected. In this process osteoblasts become entrapped in the bone matrix (as occurs in osteogenesis of osteocytic bone), but then undergo apoptosis, become mineralized and end up as part of the mineralized bone matrix.

A Novel Osteogenic Cell Line That Differentiates Into GFP-Tagged Osteocytes and Forms Mineral With a Bone-Like Lacunocanalicular Structure.

Osteocytes, the most abundant cells in bone, were once thought to be inactive, but are now known to have multifunctional roles in bone, including in mechanotransduction, regulation of osteoblast and osteoclast function and phosphate homeostasis. Because osteocytes are embedded in a mineralized matrix and are challenging to study, there is a need for new tools and cell models to understand their biology. We have generated two clonal osteogenic cell lines, OmGFP66 and OmGFP10, by immortalization of primary bone cells from mice expressing a membrane-targeted GFP driven by the Dmp1-promoter. One of these clones, OmGFP66, has unique properties compared with previous osteogenic and osteocyte cell models and forms 3-dimensional mineralized bone-like structures, containing highly dendritic GFP-positive osteocytes, embedded in clearly defined lacunae. Confocal and electron microscopy showed that structurally and morphologically, these bone-like structures resemble bone in vivo, even mimicking the lacunocanalicular ultrastructure and 3D spacing of in vivo osteocytes. In osteogenic conditions, OmGFP66 cells express alkaline phosphatase (ALP), produce a mineralized type I collagen matrix, and constitutively express the early osteocyte marker, E11/gp38. With differentiation they express osteocyte markers, Dmp1, Phex, Mepe, Fgf23, and the mature osteocyte marker, Sost. They also express RankL, Opg, and Hif1α, and show expected osteocyte responses to PTH, including downregulation of Sost, Dmp1, and Opg and upregulation of RankL and E11/gp38. Live cell imaging revealed the dynamic process by which OmGFP66 bone-like structures form, the motile properties of embedding osteocytes and the integration of osteocyte differentiation with mineralization. The OmGFP10 clone showed an osteocyte gene expression profile similar to OmGFP66, but formed less organized bone nodule-like mineral, similar to other osteogenic cell models. Not only do these cell lines provide useful new tools for mechanistic and dynamic studies of osteocyte differentiation, function, and biomineralization, but OmGFP66 cells have the unique property of modeling osteocytes in their natural bone microenvironment. © 2019 American Society for Bone and Mineral Research.

Deletion of nicotinic acetylcholine receptor alpha9 in mice resulted in altered bone structure.

Alterations in bone strength and structure were found in knockout (KO) mouse strains with deletion of several acetylcholine receptors. Interestingly, the expression of the nicotinic acetylcholine receptors (nAChR) subunit α10 was down-regulated in osteogenic differentiated mesenchymal stem cells of patients with osteoporosis whereas the expression of subunit α9 was not altered. Since nAChR subunits α9 and α10 are often combined in a functional receptor, we analyzed here the bone of adult female KO mice with single deletion of either nAChR alpha9 (α9KO) or alpha10 (α10KO). Biomechanical testing showed a significant decrease of bending stiffness and maximal breaking force in α9KO compared to their corresponding wild type mice. Furthermore, an increase in trabecular pattern factor (Tb.Pf) and structure model index (SMI) was detected by µCT in α9KO indicating reduced bone mass. On the mRNA level a decrease of Collagen 1α1 and Connexin-43 was measured by real-time RT-PCR in α9KO while no alteration of osteoclast markers was detected in either mouse strain. Using electron microcopy we observed an increase in the number of osteocytes that showed signs of degeneration and cell death in the α9KO compared to their wild type mice, while α10KO showed no differences. In conclusion, we demonstrate alterations in bone strength, structure and bio-marker expression in α9KO mice which imply the induction of osteocyte degeneration. Thus, our data suggest that nAChR containing the α9 subunit might be involved in the homeostasis of osteocytes and therefore in bone mass regulation.

Transforming growth factor-ß1 up-regulates connexin43 expression in osteocytes via canonical Smad-dependent signaling pathway.

Connexin 43 (Cx43)-mediated gap junctional intercellular communication (GJIC) has been shown to be important in regulating multiple functions of bone cells. Transforming growth factor-ß1 (TGF-ß1) exhibited controversial effects on the expression of Cx43 in different cell types. To date, the effect of TGF-ß1 on the Cx43 expression of osteocytes is still unknown. In the present study, we detected the expression of TGF-ß1 in osteocytes and bone tissue, and then used recombinant mouse TGF-ß1 to elucidate its effect on gap junctions (GJs) of osteocytes. Our data indicated that TGF-ß1 up-regulated both mRNA and protein expression of Cx43 in osteocytes. Together with down-regulation of Cx43 expression after being treated with TGF-ß type I receptor inhibitor Repsox, we deduced that TGF-ß1 can positively regulate Cx43 expression in osteocytes. Thus we next focussed on the downstream signals of TGF-ß and found that TGF-ß1-mediated smads, Smad3 and Smad4, to translocate into nucleus. These translocated signal proteins bind to the promoter of Gja1 which was responsible for the changed expression of Cx43. The present study provides evidence that TGF-ß1 can enhance GJIC between osteocytes through up-regulating Cx43 expression and the underlying mechanism involved in the activation of Smad-dependent pathway.

Extracellular Matrix Elasticity Regulates Osteocyte Gap Junction Elongation: Involvement of Paxillin in Intracellular Signal Transduction.

BACKGROUND/AIMS: Osteocytes can sense and respond to extracellular stimuli, including biochemical factors throughout the cell body, dendritic processes, and cilia bending. However, further exploration is required of osteocyte function in response to substrate stiffness, an important passive mechanical cue at the interface between osteocytes and the extracellular matrix, and the deep bio-mechanism in osteocytes involving mechanosensing of cell behavior. METHODS: We fabricated silicon-based elastomer polydimethylsiloxane substrates with different stiffnesses but with the same surface topologies. We then seeded osteocytes onto the substrates to examine their responses. Methodologies used included scanning electron microscopy (SEM) for cell morphology, confocal laser scanning microscopy (CLSM) for protein distribution, western blot for protein levels, co-immunoprecipitation for protein interactions, and quantitative real-time polymerase chain reaction for gene expression. RESULTS: SEM images revealed that substrate stiffness induced a change in osteocyte morphology, and CLSM of F-actin staining revealed that substrate stiffness can alter the cytoskeleton. These results were accompanied by changes in focal adhesion capacity in osteocytes, determined via characterization of vinculin expression and distribution. Furthermore, on the exterior of the cell membrane, fibronectin was altered by substrate stiffness. The fibronectin then induced a change in paxillin on the inner membrane of the cell via protein-protein interaction through transmembrane processing. Paxillin led to changes in connexin 43 via protein-protein binding, thereby influencing osteocyte gap junction elongation. CONCLUSION: This process -from mechanosensing and mechanotransduction to cell function - not only indicates that the effects of mechanical factors on osteocytes can be directly sensed from the cell body, but also indicates the involvement of paxillin transduction.

The Lysosomal Protein Arylsulfatase B Is a Key Enzyme Involved in Skeletal Turnover.

Skeletal pathologies are frequently observed in lysosomal storage disorders, yet the relevance of specific lysosomal enzymes in bone remodeling cell types is poorly defined. Two lysosomal enzymes, ie, cathepsin K (Ctsk) and Acp5 (also known as tartrate-resistant acid phosphatase), have long been known as molecular marker proteins of differentiated osteoclasts. However, whereas the cysteine protease Ctsk is directly involved in the degradation of bone matrix proteins, the molecular function of Acp5 in osteoclasts is still unknown. Here we show that Acp5, in concert with Acp2 (lysosomal acid phosphatase), is required for dephosphorylation of the lysosomal mannose 6-phosphate targeting signal to promote the activity of specific lysosomal enzymes. Using an unbiased approach we identified the glycosaminoglycan-degrading enzyme arylsulfatase B (Arsb), mutated in mucopolysaccharidosis type VI (MPS-VI), as an osteoclast marker, whose activity depends on dephosphorylation by Acp2 and Acp5. Similar to Acp2/Acp5-/- mice, Arsb-deficient mice display lysosomal storage accumulation in osteoclasts, impaired osteoclast activity, and high trabecular bone mass. Of note, the most prominent lysosomal storage accumulation was observed in osteocytes from Arsb-deficient mice, yet this pathology did not impair production of sclerostin (Sost) and Fgf23. Because the influence of enzyme replacement therapy (ERT) on bone remodeling in MPS-VI is still unknown, we additionally treated Arsb-deficient mice by weekly injection of recombinant human ARSB from 12 to 24 weeks of age. We found that the high bone mass phenotype of Arsb-deficient mice and the underlying bone cell deficits were fully corrected by ERT in the trabecular compartment. Taken together, our results do not only show that the function of Acp5 in osteoclasts is linked to dephosphorylation and activation of lysosomal enzymes, they also provide an important proof-of-principle for the feasibility of ERT to correct bone cell pathologies in lysosomal storage disorders. © 2018 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.